Year 1

Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people, including trained athletes, and is the most common inherited heart defect. In this proposal, we will generate human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with HCM. The specific aims are as follow:
Specific Aim 1: Generate iPSCs from patients with HCM and healthy controls.
Specific Aim 2: Determine the extent of disease by performing molecular and functional analyses of hiPSC-CMs.
Specific Aim 3: Rescue the molecular and functional phenotypes using zinc finger nuclease (ZFN) technology.

Over the past year, we have now derived iPSCs from a 10-patient family cohort with the MYH7 mutation. Established iPSC lines from all subjects were differentiated into cardiomyocyte lineages (iPSC-CMs) using standard 3D EB differentiation protocols. We found hypertrophic iPSC-CMs exhibited features of HCM such as cellular enlargement and multi-nucleation beginning in the sixth week following induction of cardiac differentiation. We also found hypertrophic iPSC-CMs demonstrated other hallmarks of HCM including expression of atrial natriuretic factor (ANF), elevation of β-myosin/α-myosin ratio, calcineurin activation, and nuclear translocation of nuclear factor of activated T-cells (NFAT) as detected by immunostaining. Blockade of calcineurin-NFAT interaction in HCM iPSC-CMs by cyclosporin A (CsA) and FK506 reduced hypertrophy by over 40%. In the absence of inhibition, NFAT-activated mediators of hypertrophy such as GATA4 and MEF2C were found to be significantly upregulated in HCM iPSC-CMs beginning day 40 post-induction of cardiac differentiation, but not prior to this point. Taken together, these results indicate that calcineurin-NFAT signaling plays a central role in the development of the HCM phenotype as caused by the Arg663His mutation.